LktA deletion mutant of P. haemolytica

ABSTRACT

Mutants of  P. haemolytica  provide excellent safety and efficacy when used as vaccines in ruminants, for example cattle, sheep, and goats, subject to pneumonic pasteurellosis. They can be administered by a variety of routes. Especially preferred is the use in animal feeds. The mutants are not reverting and contain no foreign DNA and no introduced antibiotic resistance genes.

[0001] This application claims the benefit of co-pending provisionalapplication Serial No. 60/060,060, filed Sep. 25, 1997, which isincorporated by reference herein.

TECHNICAL FIELD OF THE INVENTION

[0002] This invention is related to the field of bacterial genetics andmore particularly to the field of respiratory pathogens of farm animals.

BACKGROUND OF THE INVENTION

[0003]P. haemolytica as a pathogen causes serious economic damage to theanimal farming industry. Vaccines which have been developed in an effortto control the disease have met with variable but limited success.Because the disease is caused in significant part by the animals' ownreaction to P. haemolytica infection, inappropriately designed vaccinesmay actually worsen the clinical condition of infected vaccinates. Thus,there is a continuing need in the art for safe and effective vaccineswhich can reduce the morbidity and/or mortality of ruminants due to P.haemolytica.

SUMMARY OF THE INVENTION

[0004] It is an object of the present invention to provide a P.haemolytica bacterium useful as a vaccine strain.

[0005] It is another object of the present invention to provide a methodof inducing immunity to pneumonic pasteurellosis in ruminants.

[0006] It is an object of the present invention to provide a vaccinestrain against pneumonic pasteurellosis.

[0007] Another object of the invention is to provide a ruminant feed.

[0008] Another object of the invention is to provide a temperaturesensitive plasmid for manipulation of P. haemolytica.

[0009] These and other objects of the invention are achieved by one ormore of the embodiments described below. One embodiment of the inventionprovides a P. haemolytica bacterium which expresses no biologicallyactive leukotoxin, expresses a form of leukotoxin molecule which inducesantibodies which specifically bind to leukotoxin, and contains noforeign DNA.

[0010] Another embodiment of the invention provides a method of inducingimmunity to pneumonic pasteurellosis in ruminants. A bacterium isadministered to a ruminant. Immunity to the bacterium is therebyinduced. The bacterium expresses no biologically active leukotoxin,expresses a form of leukotoxin molecule which induces antibodies whichspecifically bind to leukotoxin, and contains no foreign DNA.

[0011] Yet another embodiment of the invention provides a feed forruminants. The feed comprises a bacterium which expresses nobiologically active leukotoxin, expresses a form of leukotoxin moleculewhich induces antibodies which specifically bind to leukotoxin, andcontains no foreign DNA.

[0012] Even another embodiment of the invention provides a vaccine forreducing morbidity in ruminants. The vaccine comprises a P. haemolyticabacterium which expresses no biologically active leukotoxin, expresses aform of leukotoxin molecule which induces antibodies which specificallybind to leukotoxin, and contains no foreign DNA.

[0013] Still another embodiment of the invention provides a temperaturesensitive plasmid. The plasmid replicates at 30° C. but not at 40° C. inP. haemolytica. Moreover, it is of the same incompatibility group as theplasmid which has been deposited at the ATCC with Accession No. ______.

[0014] The present invention thus provides the art with tools forgenetically manipulating an agriculturally important pathogen. It alsoprovides useful mutant strains which can be used effectively to reducemorbidity among ruminants, such as cattle, sheep, and goats, due toPasteurella haemolytica.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1. Fate of temperature-sensitive plasmid in Pasteurellahemolytica after passage at 30° C. and 40° C.

[0016]FIG. 2. The Pasteurella hemolytica leukotoxin operon with 3.15 kbEcoRV fragment. lktC, acylates leukotoxin structural gene to activate;LktA, leukotoxin structural gene; lktB/D, involved in leader-independentleukotoxin export.

[0017]FIG. 3. In-frame deletion of 3.15 kb EcoRV fragment of lktCA usingNaeI.

[0018]FIG. 4. Integration of replacement plasmid into chromosome.

[0019]FIG. 5. Resolution of replacement plasmid from chromosome.

[0020]FIG. 6. Western blot of native leukotoxin and ΔlktA using anti Lktmonoclonal antibody.

DETAILED DESCRIPTION

[0021] It is a discovery of the present invention that a mutant form ofleukotoxin is useful as a vaccine. The mutant form is made by anon-reverting mutant of P. haemolytica. Moreover, this mutant form hasbeen found to be useful when administered to the tonsils, via the oralroute, and via the nasal route. Thus extremely inexpensive and easymethods of vaccinating animals can be accomplished, simply by topdressing animal feed.

[0022] The mutant preferably is a deletion mutant. One such mutantleukotoxin protein made is about 66 kD, although other such mutants canbe used, so long as the protein is long enough to be immunogenic,preferably at least 10, 15, or 20 amino acids long. It is believed thata longer deleted molecule is preferred to achieve a strong immuneresponse. It is preferred that the mutant bacterium which makes theleukotoxin contains no exogenous genes, such as drug resistance genes,which can cause environmental and health problems if not contained. Inaddition, it is preferred that the mutation be a non-reverting mutation,such as a deletion mutation.

[0023] Mutant forms of leukotoxin of the present invention induceantibodies which specifically bind to leukotoxin. Antibodies whichspecifically bind to leukotoxin provide a detection signal at least 2-,5-, 10-, or 20-fold higher than a detection signal provided withproteins other than leukotoxin when used in Western blots or otherimmunochemical assays. Preferably, antibodies which specifically bind toleukotoxin do not detect other proteins in immunochemical assays and canimmunoprecipitate leukotoxin from solution. More preferably, theantibodies can be detected in an indirect hemagglutination assay and canneutralize leukotoxin.

[0024] Although the oral route is preferred for ease of delivery, otherroutes for vaccination can also be used. These include withoutlimitation, subcutaneous, intramuscular, intravenous, intradermal,intranasal, intrabronchial, etc. The vaccine can be given alone or as acomponent of a polyvalent vaccine, i.e., in combination with othervaccines. Bacteria can be used in the vaccine to supply the mutantleukotoxin protein. The bacteria in the vaccine formulation can be live,lyophilized, lyophilized and reconstititued, or killed. Moreover,bacterial lysates, extracts or culture supernatants which contain theLtkA deletion protein can be used in the vaccine formulation. Purifiedprotein can also be used, if desired. It can be formulated with otherimmunogenic proteins.

[0025] Also provided by the present invention is a temperature sensitiveplasmid which replicates at 30° C. but not at 40° C. in P. haemolytica.Preferably the plasmid is of the same incompatibility group as pD80, ie., it shares the same origin of replication. One such plasmid has beendeposited at the ATCC with Accession No. ______.

[0026] Vaccination with modified-live combination of P. haemolyticaserotypes 5 and 6 protects against combined homologous virulentchallenge extremely well, based on clinical signs, postmortem lesions,and results of bacterial culture. Animals which have been so vaccinatedremain active, alert, afebrile, and on-feed. The vaccine can not onlyprevent death due to P. haemolytica, but can also reduce symptoms ofpneumonic pasteurellosis, such as lung lesion volume, fever, decreasedappetite, loss of lung ventilation capacity, fibrinous pleural effusionand/or adhesions, bacterial load, and depression.

[0027] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLE 1 Modified-live Oral and Parenteral Vaccines AgainstShipping-fever and Pneumonic Pasteurellosis of Cattle, Sheep, and GoatsBased on In-frame Clean Deletions of the Leukotoxin Structural Gene ofPasteurella haemolytica Materials and Methods

[0028] Mutagenesis of plasmid origin of replication. A 1.2 kb DNAfragment containing the putative pD80 origin of replication wasamplified by PCR using the 4.3 kb ampicillin-resistance plasmid isolatedfrom P. haemolytica serotype 1 strain NADC-D80 as template (1). Forwardprimer 5′-CCG GAT CCC CAA TTC GTA GAG GTT TC-3′ (SEQ ID NO:1) andreverse primer 5′-CCG GAT CCG CTG AAA GCG GTC GGG GG-3′ (SEQ ID NO:2)were used. The product was cloned into pCR2.1 vector (Invitrogen, SanDiego Calif.) using the manufacturer's directions. A kanamycin-cassettewas prepared by passage of pBC SK (Stratagene, La Jolla Calif.)containing a derivative of the Tn903 kanamycin gene (Pharmacia Biotech,Piscataway N.J.) cloned into the unique EcoR1 site through the E. colistrain PhaImtase to protect against PhaI-cleavage.

[0029] The cloned 1.2 kb insert (1 μg) was excised from pCR2.1 by EcoR1digestion and ligated overnight to the EcoR1-digested PhaI-methylatedkanamycin cassette (0.25 μg). The ligation mixture was concentrated byEtOH precipitation and electroporated (Gene-pulser, Bio-Rad, RedfieldCalif.) into P. haemolytica serotype 1 strain NADC-D153 using 18 kv/cmand 1000 W.

[0030] Plasmid DNA was obtained from a kanamycin-resistant transformantwhich was 2.5 kb in size and cleaved into fragments of 1.2 and 1.3 kbwhen subjected to EcoR1. One μg of the plasmid was mutagenized withhydroxylamine for 1 hour at 65° C. as previously described (2).

[0031] Selection of temperature-sensitive plasmid origin of replication.The mutagenized plasmid was dialyzed overnight at 4° C. against TE,concentrated by ethanol precipitation, and electroporated into freshNADC-D153 as described above. After 2 h recovery in Columbia broth(Difco Laboratories, Detroit Mich.), the cells were plated onto 10Columbia blood agar base plates containing 50 μg/ml kanamycin and wereincubated at 30° C. After 20 h, the plates were moved to 40° C. for anadditional 6 h.

[0032] Colonies were selected which were atypically small and cloned tofresh kanamycin plates and were incubated overnight at 30° C. Growthfrom the clones was duplicated onto plates with and without kanamycinand incubated overnight at 40° C.

[0033] Clones which failed to grow on selective media at 40° C. but grewwithout selection were presumed to be temperature-sensitive for eitherplasmid maintenance or for expression of kanamycin. Growth from plateswithout antibiotic selection at 40° C. was passed to selective plateswhich were incubated overnight at 30° C. Clones which exhibited light orno growth on this passage were presumed to contain temperature-sensitiveorigins of replication. These clones were passed from the original 30°C. selective plate to fresh selective plates and to selective broth. Theclones were rechecked by passage with and without selection at 40° C.and 30° C. Although each clone exhibited the correct phenotype, plasmidmini-preps from the broth cultures yielded small amounts of a 2.5 kbplasmid from only one of the cultures. The remaining clones were notexamined further.

[0034] Construction of dual-origin temperature-sensitive shuttle vector.The temperature-sensitive origin of replication was excised from theabove plasmid by EcoR1 digestion and then made blunt by treatment withKlenow fragment of DNA polymerase I and all four dNTPs. The fragment wasligated overnight at 4° C. with SmaI-digested pBC SK. The ligation mixwas used to transform E. coli DH10-B (Life Technologies, GaithersburgMd.).

[0035] Plasmid containing a 1.2 kb insert was recovered from achloramphenicol-resistant colony. The plasmid was digested with SalI andligated to SalI-digested kanamycin cassette overnight at 4° C. Theligation mixture was electroporated into E. coli DH10-B and plated ontokanamycin 50 μg/ml. Plasmid recovered from a kanamycin-resistant colonywas digested with BssHII, made blunt as above, treated with calfalkaline phosphatase to remove the terminal phosphates, and ligatedovernight at 4° C. with an approximately 900 bp blunt fragmentcontaining the ColE1 plasmid origin of replication.

[0036] The ligation mixture was electroporated into E. coli PhaImtase(1) and plated onto kanamycin-containing plates. Plasmid was recoveredfrom a kanamycin-resistant colony which yielded a single approximately3.5 kb fragment with EcoR1-digestion and fragments of 2.2 and 1.3 kbwith SalI-digestion. The plasmid was given the designation pBB80C. Theplasmid was electroporated into P. haemolytica to confirm thetemperature-sensitive origin of replication; it still supportedbacterial growth on kanamycin at 30° C. but not at 40° C.

[0037] Cloning and manipulation of lktA. A 3.15 kb EcoRV fragment of P.haemolytica genomic DNA containing lktC and approximately 75% of thelktA coding region was ligated into the EcoRV site of pBB80C. Theresulting plasmid was amplified in E. coli DH10-B and given thedesignation pBB80ClktA.

[0038] Plasmid pBC SK (0.25 μg, used to provide additional NaeI-sites intrans) was mixed with 0.25 μg pBB80ClktA and digested with NaeI for 18 hat 37° C. The resulting partially digested plasmid DNA was extractedwith phenol-chloroform-isoamyl alcohol (PCI), precipitated with ethanol,ligated at 4° C. overnight, then re-extracted and precipitated. Theligation mixture was digested with PvuII, which cleaves both pBC SK andthe 1035 bp NaeI fragment internal to LktA.

[0039] Five ng of the digested DNA was electroporated into E. coliPhaImtase and plated on Columbia blood agar base plates containing 50μg/ml kanamycin. Plasmid DNA from selected transformants was screened bydigestion of plasmid minipreps with EcoRV alone or together with NgoM1(an isoschizomer of NaeI). A clone containing a 1035 bp deletion wasselected and given the designation pBB80CΔlktA.

[0040] Recovery of leukotoxin mutants. Plasmid pBB80CΔlktA waselectroporated into fresh P. haemolytica strain NADC-D153 at 18 kv/cmand 1000 W. The cells were allowed to recover 2 hours at 30° C. in 1 mlColumbia broth, then were plated 100 μl/plate on Columbia agar platescontaining 50 μg/ml kanamycin. After 48 h incubation at 30° C., fourcolonies were passed to kanamycin plates containing 5% defibrinatedbovine blood and were incubated overnight at 37° C. to select forsingle-crossover mutants. Four colonies from the 37° C. passage, 2hemolytic and 2 non-hemolytic from each original transformant (16total), were passed to Columbia broth without selection and incubatedovernight at 30° C. to resolve the single-crossover mutations.

[0041] Growth from the 30° C. Columbia broth was struck for isolationonto blood agar base plates containing 5% defibrinated bovine blood andincubated overnight at 37° C. Growth was also passed to fresh Columbiabroth successively for a total of 4 passages at 30° C. to furtherascertain the rate at which kanamycin resistance was lost at 30° C.without selection. Isolated colonies from the first 30° C. passage wereduplicated in an array on selective and on non-selective platescontaining 5% defibrinated bovine blood.

[0042] A kanamycin-sensitive clone which demonstrated no detectablehemolytic activity on the non-selective plate was selected for furtherstudy. Additional strains of P. haemolytica obtained from the repositoryat the National Animal Disease Center were later subjected to similartreatment as above. These strains were isolated from pneumonic lung andincluded: NADC D632, ovine serotype 1; NADC D121, ovine serotype 2; NADCD110, ovine serotype 5; NADC D174, bovine serotype 6; NADC D102, ovineserotype 7; NADC D844, ovine serotype 8; NADC D122, ovine serotype 9;and NADC D712, ovine serotype 12.

[0043] Characterization of the putative leukotoxin mutant. To define thechromosomal deletion, DNA was amplified from whole cells of the putativeleukotoxin mutant and from its parent strain NADC-D 153 by PCR, usingprimers nested within the EcoRV termini of the original 3.15 kb EcoRVgenomic fragment. The products were electrophoresed on a 1.2% agarosegel both intact and after NgoM1 digestion.

[0044] To determine leukotoxic activity, log-phase culture supernatantsfrom the putative mutant and its parent were prepared from Columbiabroth 3 hour cultures. Two-fold dilutions of the supernatants wereassayed using BL-3 target cells and MTT dye (7).

[0045] To determine the expression of the putative altered leukotoxinproduct, the culture supernatants, as well as a third culturesupernatant from our original leukotoxin deletion mutant which producesno detectable leukotoxin, were concentrated approximately 15-fold using30,000 mw ultrafilters (Centriprep, Amicon, Beverly, Mass.). Theretentate was electrophoresed in duplicate on SDS-PAGE, and one gel wasstained using Coomasie blue.

[0046] The second gel was blotted onto a nylon membrane for Western blotanalysis. The membrane was washed, probed with anti-leukotoxinmonoclonal antibody 601 (provided by Dr. S. Srikumaran, Lincoln Nebr.),labeled with anti-mouse IgG alkaline phosphatase-conjugated antibody(Sigma), and stained with nitro blue tetrazolium (Sigma).

[0047]Pasteurella haemolytica mutants of other than NADC D153ΔlktAserotype 1 were characterized by PCR analysis and growth characteristicson blood agar plates only. Their production of altered leukotoxinprotein was not confirmed.

Results

[0048] The temperature-sensitive origin of replication derived from theendogenous P. haemolytica ampicillin-resistance plasmid proved to be auseful tool for the construction of deletion mutants in that organism.The origin of replication was assumed to reside within a non-codingregion from nucleotides 3104 to 4293 of the native plasmid. The 1.2 kbPCR product of that region ligated to a 1.3 kb Tn903 kanamycin cassetteresulted in a 2.5 kb product capable of the stable transformation of P.haemolytica as evidenced by less than 1% loss of plasmid after 100generations in broth culture at 37° C. These data indicate thatessential replication functions reside within that 1.2 kb region of thenative plasmid.

[0049] Efficiency of transformation of P. haemolytica dropped about10-fold after hydroxylamine mutagenesis, indicating perhaps the DNA wasnot particularly damaged. Nevertheless, 10 colonies which wereatypically small were recovered after 20 hours at 30° C. and 6 hours at40° C. Two of these colonies grew on selection at 40° C. and werediscarded. Of the remaining 8 colonies, four were found to retainkanamycin-resistance after passage without selection at 40° C. These 4colonies were presumed to contain plasmid which was temperaturesensitive for the expression of kanamycin-resistance and were alsodiscarded.

[0050] The remaining 4 colonies, presumed to contain plasmid which wastemperature sensitive for maintenance, were recovered from the original30° C. plate and passed again to 40° C. and 30° C. Although each grewwell without selection at both temperatures, failed to grow withselection at 40° C., and failed to retain kanamycin-resistance after 40°C. passage, only one clone yielded sufficient plasmid for further studyby a rapid alkaline lysis procedure. It was assumed the other 3 coloniesalso contained plasmid, but the rapid plasmid purification procedurefailed to recover sufficient quantities to visualize on agarose gels.The plasmid yield from the positive clone was very low.

[0051] To facilitate subsequent isolation and cloning, amultiple-cloning site and a ColE1 origin of replication was added to thetemperature-sensitive pD80 origin. The temperature-sensitive origin anda fresh kanamycin cassette were placed within the multiple-cloning siteof pBC-SK, then the vector backbone was replaced with a <1 kb copy ofthe ColE1 origin. This approximately 3.5 kb plasmid, pBB80C, retainsmost of the unique restriction sites of pBC-SK, replicates efficientlyin E. coli, and transforms P. haemolytica at 30° C. with moderateefficiency. In P. haemolytica the ColE1 origin fails to supportreplication, and plasmid maintenance is dependent on the mutated pD80origin. In this situation, pBB80C failed to support growth on selectivemedia at both 37 and 40° C. but supported moderate growth at 30° C.(FIG. 1).

[0052] To introduce an in-frame deletion within the coding region of P.haemolytica lktA by allelic exchange, an EcoRV fragment containing partof the leukotoxin operon was cloned into pBB80C, yielding pBB80ClktA.The clone extended approximately 500 bp upstream from the lktC startcodon and included about 75% of lktA (FIG. 2).

[0053] Within the EcoRV fragment are two NaeI sites which cleave betweencodons within lktA leaving blunt termini (FIG. 3). The NaeI sites aresituated nearly evenly 1 kb apart within the EcoRV fragment. Digestionof pBB80ClktA with NaeI was complicated by the fact that NaeI is among agroup of restriction endonucleases which show a dramatic site preferencefor cleavage (4). This enzyme requires simultaneous interaction with twocopies of its recognition sequence before cleaving DNA. With certainenzymes of this type, the second copy may be supplied in trans, so itwas chosen in this experiment to supply additional recognition sites tothe digestion misture by adding pBC SK, which contains one site.Although this strategy resulted in incomplete cleavage after overnightdigestion, a 1 kb fragment was evident in the mixture, indicating bothNaeI sites had cleaved on some of the pBB80ClktA molecules.

[0054] Cleavage after ligation with PvuII, which is contained both inpBC SK and within the 1 kb NaeI fragment to be deleted, apparentlyeliminated most undesired products, because all transformants screenedfor pBB80CΔlktA contained the desired 1035 bp deletion. Each of theserecleaved with NgoM1, indicating the new NaeI site was intact and theproduct should be in-frame to the lktA start codon.

[0055]Pasteurella haemolytica transformed with pBB80CΔlktA requirednearly 48 hours to achieve good colony size at 30° C. Passage to 37° C.by simply streaking heavily on a kanamycin-containing plate resulted innumerous isolated colonies, some hemolytic and some not. These resultsare consistent with specific integration of the plasmid into theleukotoxin operon (FIG. 4). Since the replacement plasmid containedintact operon sequence upstream from the deletion, including thepromotor, upstream single-crossover products were expected to expressthe entire operon normally. Downstream single-crossover products,however, were expected to contain two defective copies of lktA, sincethe C-terminal encoding 25% of lktA was not present on the replacementplasmid. One copy of the leukotoxin gene therefore would be expected tocontain the 1 kb deletion and the other copy a truncated C-terminus.Hemolytic activity has previously been shown to be correlated withexpression of active LktA (3, 5, 6).

[0056] Passage of single-crossover products at 30° C. resulted in anunexpectedly low rate of plasmid resolution from chromosome. Previouswork with pBB 192C, a temperature-conditional plasmid derived from theP. haemolytica streptomycin-resistance plasmid, exhibited 90 to 99%reversion to kanamycin sensitivity after a single passage at 37 or 30°C. respectively. In this experiment, of 80 isolated colonies testedafter one passage at 30° C., only two became sensitive to kanamycin. Oneof the two was non-hemolytic and was later shown to be adouble-crossover mutant (FIG. 5). Further passage increased thepercentage of kanamycin-sensitive CFU in non-selective cultures tonearly 50% after 4 passages. Many of these colonies exhibited anon-hemolytic phenotype and were probably double-crossover products.

[0057] To generate mutants of the other serotypes, 4-8 hemolyticsingle-crossover products were selected and passed at 30° C. for one ormore passages in broth. Growth was struck for isolation on each passage,and non-hemolytic colonies were selected for testing by PCR and growthon kanamycin-containing media. In each case, non-hemolytic colonieswhich were kanamycin-sensitive were confirmed by PCR to be deletionmutants containing single NaeI sites.

[0058] We assume that pBB192C contains a more robust origin ofreplication than does pBB80C, as evidenced by the relative amounts ofplasmid recovered from the respective cultures. If activity of anintegrated plasmid origin destabilizes chromosomal replication, it wouldbe expected that greater instability would be realized as plasmid originactivity increases. This could account both for greater resolving ratesof pBB192C at 30° C. than at 37° C. and for the lower rates of resolvingof pBB80C compared to pBB192. During construction of our firstleukotoxin deletion mutant, a large number of single crossover productswere obtained using suicide replacement plasmid (3), which containedampicillin selection. Although both the homologous arms were similar inlength to those of the current experiment, passage for even 100generations resulted in no reversion to a hemolytic phenotype or loss ofampicillin-resistance. These data further indicate that it is theactivity of plasmid origin which destabilizes the single-crossoverproducts.

[0059] PCR products from the putative leukotoxin mutants and theirparent strains were found to be 2 kb and 3 kb in size respectively,indicating a deletion had been introduced into their respective lktA.Digestion of the PCR products with NgoM1 revealed 2 bands ofapproximately 1 kb from the mutants and 3 bands of approximately 1 kbfrom the parent strains, indicating the deletions should be in-frame toLktA. Leukotoxin activity in culture supernatants against BL-3 targetcells from the serotype 1 mutant was <1:2 compared to 1:1024 from theparent strain, indicating no detectable activity.

[0060] A new protein of approximately 65 kDa was detected in the culturesupernatant of this mutant by SDS-PAGE, consistent with the predictedmolecular weight of the deleted product. By Coomasie staining, the newproduct exceeded the concentration of the native LktA protein producedby the parent strain grown and harvested alongside the mutant. Thesmaller size of this product may allow more rapid or economicalexpression of the gene. The product reacted with the neutralizingmonoclonal antibody 601 at an apparent molecular weight of 66 kDa (FIG.6). No reaction was observed at 101-104 kDa, the apparent molecularweight of the native product observed in the culture supernatant of theparent strain.

EXAMPLE 2 Assessment of Vaccine Efficacy in Small Ruminants afterIntramuscular Injection of a Polyvalent Combination of P. haemolyticaSerotypes 5 and 6 Materials and Methods

[0061] Vaccination of animals. Four lambs (Columbia, approximately 25kg) and six goats (Toggenburg, approximately 15 kg) were colostrumdeprived and raised at the National Animal Disease Center, Ames, Iowa.Two lambs and three goats were randomly selected and vaccinated with4×10⁷ CFU each of P. haemolytica NADC D110ΔlktA and NADC D174ΔlktA(serotypes 5 and 6 respectively) in 1 ml Earles Balanced Salt Solution(EBSS), pH 7.4. The suspension was delivered intramuscularly in themid-cervical region. After three weeks, the animals were similarlyrevaccinated.

[0062] Ten days after the second vaccination all ten animals werechallenged with 8.5×10⁷ CFU each of the parent strains NADC D110 andNADC D174 mixed in a total volume of 5 ml EBSS instilled intratracheallyat the tracheal bifurcation with a catheter. The inoculum was chasedwith 5 ml sterile EBSS. Five days after challenge all surviving animalswere euthanized and necropsied.

[0063] Bacteria. Pasteurella haemolytica strains NADC D110 (serotype 5,ovine lung isolate) and NADC D174 (serotype 6, bovine lung isolate) weregrown separately in Columbia broth (Difco Laboratories, Detroit Mich.)approximately 3 hours to late log phase, about 2×10⁹ CFU/ml. Growth wasdiluted in EBSS 1:50 for the vaccine dose or 1:100 for the challengedose. The two strains were mixed in equal volume and kept on ice priorto animal inoculation.

[0064] Samples and data collection. Sera were collected the day of thefirst vaccination, 2 weeks later, the day of challenge exposure, and theday of necropsy. Rectal temperatures were recorded for 3 days after eachvaccination and twice daily from challenge exposure to necropsy.Clinical scores were subjectively assessed on the same schedule asrectal temperatures, based on degree of depression and appetite. Atnecropsy, lung specimens from 1 to 3 grams in weight were obtained fromareas containing abnormalities, when possible, for bacterialenumeration. Swab specimens were obtained from trachea, kidney, andliver for bacterial isolation. Lung lesion volumes were estimated foreach lobe of the lung, including both consolidated areas and those whichappeared merely atelectic. Total lung lesion scores were expressed as apercentage where each lobe was adjusted for an approximation of itscontribution to air exchange as follows: right cranial lobe, 6%; rightcranial half of the middle lobe, 5%; right caudal half of the middlelobe, 7%; right caudal lobe, 35%; accessory lobe, 4%; left cranial lobe,4%; left middle lobe, 6%; and left caudal lobe, 32%.

[0065] Sample processing. Sera were tested for P. haemolytica antibodyby indirect hemagglutination (IHA) against serotypes 5 and 6 (allanimals) and by leukotoxin neutralization (vaccinates only) using BL-3cells and MTT dye (7, 8). Lung specimens were weighed, and EBSS wasadded to bring the tissue plus fluid volume to 10 times the weight. Thespecimens were ground to yield a homogenous suspension, and ten-folddilutions were made in EBSS.

[0066] The dilutions (100 μl) were spread onto blood agar base platescontaining 5% defibrinated bovine blood and incubated overnight at 37°C. Colonies exhibiting typical P. haemolytica morphology wereenumerated, and 20 representative colonies (where available) wereserotyped using specific antisera (9). Swabs were rolled onto one-thirdof fresh blood agar plates and then each side of a sterile loop was usedto semi-quantitatively streak for isolation onto the remaining thirdsconsecutively.

Results

[0067] No local reaction was palpable or visible following eithervaccination in any vaccinate. The first dose of vaccine elicited afebrile response, particularly in the sheep, which had a fever on day 2and 3 which peaked at 40.3° C. on day 3. The second injection elicitedno clinical response.

[0068] Prior to vaccination, the animals had a low IHA titer againstboth serotypes 5 and 6 of P. haemolytica (Table 1). After the firstvaccination, the vaccinates' titer increased over 8-fold against bothserotypes. No response was evident after the second dosage. Only aslight increase in antibody titer, about 50%, occurred after challengeexposure. The control animals' titer increased slightly, about double,prior to challenge exposure. Between the time of challenge exposure andnecropsy, the one surviving control sheep increased its titer againstboth serotypes by about 32-fold.

[0069] Leukotoxin neutralization titers in the vaccinates increasedvariably. Both lambs and two goats seroconverted (increased at least4-fold) after the first vaccination; one of the animals alsoseroconverted to the second dose. One goat remained seronegativethroughout the study.

[0070] Following challenge, none of the vaccinates had a fever at anytime. They remained alert and eating all their food until necropsy. Thecontrol animals had a fever the day after exposure averaging 40.7° C.All control goats and 1 control sheep died overnight between the firstand second day after exposure. The remaining control sheep remainedfebrile, anorexic, and depressed until necropsy.

[0071] Inspection of the vaccine injection site at necropsy revealed nodetectable reaction in the muscle. Slight subcutaneous discolorationabout 1 cm in diameter due to hemorrhage was detected in both sheep andtwo of the three goats.

[0072] Lung lesion volume of the vaccinates, corrected for ventilationcapacity of each lobe, averaged 3.5% (Table 2). One goat had 95% of itsaccessory lobe with moderately firm consolidation from which 1.3×10⁶CFU/g (equally of serotypes 5 and 6) were recovered. The remaining lunglesions were soft, consistent with atelectasis.

[0073] Of 19 lung specimens quantitatively cultured, 5 yielded P.haemolytica. Two animals yielded no P. haemolytica from their lung. Twoyielded from 2×10³ to 7×10³ CFU/g from their right cranial lobes orcranial half of the middle lobe only. The animal with accessory lobeinvolvement also yielded 1×10³ CFU/g from the right caudal half of itsmiddle lobe and moderate growth from its tracheal swab. All othertracheal swabs from vaccinates were culture negative, as were swabs fromliver and kidney.

[0074] One sheep had tight adhesions of visceral to parietal pleura andto the pericardium ventrally on both right and left sides involving alllobes. This sheep contained only minor lesions of atelectasis andyielded only 2×10³ CFU/g from its right cranial lobe; both other lobescultured were negative.

[0075] Lung lesion volume of the controls (corrected for ventilationcapacity of each lobe) averaged 52% (Table 2). The four animals whichdied contained large amounts of fibrinous pleural effusion and fibrinouspleural adhesions. The lung lesions were firm or moderately firm, andemphysematous and/or crepitous areas were evident. The sheep whichsurvived until the time of necropsy contained about 100 cc pleuraleffusion and a large (about 250 cc) fibrous mass occupying the plerualspace over the right cranial and middle lobes.

[0076] The lung lesions consisted primarily of firm fibrinousconsolidation in this animal. Of 17 cultured lung specimens, all yieldedP. haemolytica from as few as 2.5×10⁴ CFU/g to 4×10⁹ CFU/g. Thegeometric mean count for the four animals which died acutely was 2.5×10⁸CFU/g; the surviving sheep had a mean count of 2.5×10⁵ CFU/g. Trachealswabs from the four animals which died yielded heavy growth of P.haemolytica. The surviving sheep yielded light growth from its trachea.Liver swabs of all four and kidney swabs of two of the animals whichdied yielded P. haemolytica. The surviving sheep was culture negative inboth liver and kidney.

[0077] Serotyping of isolates from lung revealed that the few coloniesrecovered from vaccinates were of serotype 5, except for the activelyinfected accessory lobe of one goat which yielded equal amounts of bothserotype 5 and 6. Control animals tended to yield a mixture of serotypesfrom each lobe, but the mixture varied widely from lobe to lobe in theanimals which died acutely (e.g. 95% of serotype 5 in the right craniallobe to only 5% of serotype 5 in the right caudal lobe). Isolatesrecovered from kidney or liver tended to be homogenous with respect toserotype in any given animal, but two animals contained serotype 5 inthese tissues, and the other two contained serotype 6.

[0078] The first dose of vaccine can induce a febrile response. The lackof a febrile response and immune response from the second dose impliesthat substantial immunity is conferred by the first dose. The seconddose was apparently quickly dealt with by the immune system and did notdevelop sufficient antigenic mass to elicit an anemnestic response. Thedosage of organisms delivered in the vaccine (nearly 10⁸ CFU) may haveexceeded that necessary to confer sufficient immunity. Modified-livevaccines typically would be delivered at a lower dose, perhaps 10⁵ to10⁷ CFU. The failure of the second dosage of vaccine to stimulatefurther antibody, as measured by IHA, may indicate that two doses wereunnecessary and that a single dose would have been sufficient.

[0079] The reactions observed at the vaccine injection sites wereextremely minor and did not involve muscular tissue, consistent withfindings using leukotoxin negative mutants of serotype 1 in cattle. Thiscontrasts greatly with the response of leukotoxin positive strains givenintramuscularly to cattle, which evidence large swellings and necrosisin the area, often opening through the overlying skin. It is likely thatlittle or no local adverse reaction would occur with subcutaneous orintradermal vaccination, an alternative that may also tend to reduce thefebrile response to vaccination.

[0080] Thus polyvalent intramuscular vaccine elicited marked immunity insheep and goats against polyvalent challenge. Adverse reactions werelimited to a febrile response after injection which might be controlledby reduced vaccine dosage or an alternative route of administration.

EXAMPLE 3 Assessment of Vaccine Efficacy in Cattle after OralAdministration and after Intramuscular Injection Materials and Methods

[0081] Vaccination of animals. Sixteen dairy-type calves, approximately150 kg, were obtained from a local dairy and housed at the NationalAnimal Disease Center, Ames, Iowa. The calves were randomly assigned toone control group of six and two vaccinate groups of 5. Each group wasseparately housed under similar conditions to prevent spread of vaccineorganism between groups.

[0082] To each calf in one group of vaccinates was subcutaneouslyadministered in the mid cervical region 1 ml of EBSS containing 1×10⁷CFU P. haemolytica serotype 1, NADC D153ΔlktA in-frame deletion mutanton day 0. These calves were revaccinated similarly with 7.0×10⁶ CFU in 1ml EBSS on day 21. The other group of vaccinates was fed a pelletedration (Growena, Ralston Purina, St. Louis Mo.) onto which 50 ml totalvolume of a fresh broth culture containing 1×10⁹ CFU/ml NADC D153ΔlktAin-frame deletion mutant was poured on day 0. The calves were similarlyfed 50 ml of 7×10⁸ CFU/ml on day 21.

[0083] On day 28 all calves were challenged intratracheally with 25 mlof the parent P. haemolytica in EBSS at 2×10⁷ CFU/ml using a catheterplaced at the tracheal bifurcation. The challenge was chased with 25 mlsterile EBSS. Calves which survived challenge were euthanized 4 or 5days after challenge and necropsied.

[0084] Bacteria. Pasteurella haemoltica strain NADC D153 and itsleukotoxin mutant were grown in Columbia broth approximately 2.5 hoursto mid log phase, about 1×10⁹ CFU/ml. Growth was diluted 100-fold inEBSS for injection or 50-fold for challenge. Growth was used unwashedand undiluted for oral administration. All preparations were kept on iceprior to animal inoculation.

[0085] Samples and data collection. Sera were collected 3 days prior tothe day of the first vaccination, 3 weeks later, the day of challengeexposure, and the day of necropsy. Rectal temperatures were recorded for3 days after each vaccination and twice daily from challenge exposure tonecropsy. Clinical scores were subjectively assessed on the sameschedule as rectal temperatures, based on degree of depression andappetite.

[0086] At necropsy, lung speciments were obtained and treated asdescribed in Example 2, above.

[0087] Sample processing. Sera were tested for P. haemolytica antibodyby IHA against serotype 1 and by leukotoxin neutralization using BL-3cells and MTT dye. Lung specimens were weighed, and EBSS was added tobring the tissue plus fluid volume to 10 times the weight. The specimenswere ground to yield a homogenous suspension, and ten-fold dilutionswere made in EBSS. The dilutions (100 ml) were spread onto blood agarbase plates containing 5% defibrinated bovine blood which were incubatedovernight at 37° C. Colonies exhibiting typical P. haemolyticamorphology were enumerated and, where available, 10 representativecolonies were serotyped using specific antisera. Swabs were rolled ontoone-half of fresh blood agar plates, and then each side of a sterileloop was used to semi-quantitatively streak for isolation onto theremaining two quarters consecutively

Results

[0088] No local reaction was palpable or visible following eitherparenteral vaccination. None of the calves exhibited a febrile responseafter the first parenteral or oral vaccination. One parenterallyvaccinated calf exhibited a transient (1 day) fever of 40.4° C. afterthe second dose; no adverse reaction was noted with any of the remainingcalves.

[0089] Prior to vaccination, the animals had a low IHA titer againstserotype 1 P. haemolytica (Table 3). After the first vaccination, theantibody titer in calves fed the vaccine increased at least 8-fold overtheir prevaccination titers. The second oral dose did not increase, andin some cases titers dropped 2-fold. Titers of parenterally vaccinatedcalves increased only about 2-fold after the first dose of vaccine,during which time similar titer increases occurred in the controlcalves. The second dose of parenteral vaccine elicited additionalantibody response in the parenteral vaccinates, seroconverting (4-foldincrease) 3 of these 5 calves.

[0090] Leukotoxin neutralization titers were relatively high in mostcalves prior to vaccination (Table 3). Two orally vaccinated calvesseroconverted after the first vaccine dose. One parenterally vaccinatedcalf seroconverted after the second vaccine dose. Overall,antileukotoxin titers increased in both vaccinated groups on successivebleedings. Antileukotoxin titers of control calves tended to decrease onsuccessive bleedings.

[0091] Following challenge, some but not all of the parenteralvaccinates exhibited fevers under 41° C.; the oral vaccinates remainedafebrile. All the vaccinates remained alert and on-feed. One controlanimal died the third day after challenge. Another was euthanized on day3 nearly moribund. Two of the remaining control calves were depressedand off-feed and maintained a fever until euthanasia on day 4 or 5. Oneof these calves was recumbent and thumping at the time of euthanasia.The remaining 2 control calves became afebrile the third day afterchallenge. They resumed eating and were deemed alert.

[0092] Lung lesion volume, corrected for ventilation capacity of eachlobe, averaged 4.4% for orally vaccinated animals, 7% for thosesubcutaneously vaccinated, and 32% for unvaccinated controls (Table 4).Lung lesions of both vaccinated groups were predominantly soft,consistent with atelectasis. Localized areas of firm consolidation werenoted in 2 of the orally vaccinated calves and 4 of the parenteralvaccinates, with limited pleuritis and moderate pleural adhesions in twoanimals of each group. These firm areas were confined to fractions ofsingle lung lobes in each case. Unvaccinated controls had multiple lunglobes which contained a substantially higher percentage involvement withfirm, fibrinous consolidation associated with edema and extensivefibrinous pleuritis. Three of the six control animals contained a largeamount of pleural effusion.

[0093] Bacterial culture of lung specimens showed that 2 orallyvaccinated calves and 1 parenterally vaccinated calf were culturenegative in all tested lobes. The remaining vaccinates tended to haveone or two specimens which yielded substantial amounts of P.haemolytica, up to 5×10⁷ CFU/g. The remaining lobes were either culturenegative or contained low amounts of P. haemolytica, about 10³ CFU/g.Unvaccinated control animals yielded multiple specimens with highnumbers of P. haemolytica, over 10⁷ CFU/g with many between 10⁹ and 10¹⁰CFU/g. Nasal swabs yielded P. haemolytica from 1 parenterally vaccinatedcalf and 4 control calves. Tracheal swabs were culture-positive for P.haemolytica in 4 control calves, 1 of which was nasal culture-negative.Pleural fluid was culture positive in 3 control calves. All vaccinateswere culture-negative from trachea and pleural fluid No P. haemolyticawere recovered from liver or kidney of any calf. All P. haemolytica wereβ-hemolytic, and those tested were serotype 1.

[0094] Thus, vaccination with the modified-live P. haemolytica protectedagainst virulent challenge, whether the vaccine was administeredsubcutaneously or orally after top-dressing feed.

[0095] Adverse reactions to vaccination were limited to one animalexhibiting a transient fever after the second subcutaneous injection ofvaccine. No local irritation or swelling was evident nor any postmortemabnormalities at the injection site, and no clinical abnormalities werenoted in any animal, whether injected or fed vaccine. The vaccine dosageused for injection was about 4-fold lower than that used in Example 2,above.

[0096] Seroconversion by IHA was impressive for animals orallyvaccinated. All animals' titer increased at least 8-fold after the firstexposure. Less impressive was seroconversion after subcutaneousinjection. No animals seroconverted after the first dose, and only 3 of5 sevoconverted after the second dose. The IHA procedure has been founduseful as a measure for animals' prior experience with P. haemolytica ofspecific serotypes (10-13). Its utility for predicting resistance todisease is unclear, however (14-16). While some researchers find acorrelation between IHA titers and disease, others find none. If oneassumes that the serotype-specific antigens employed in the IHAprocedure are not those involved in humoral protection, the discrepencycan be explained. Vaccination could elicit an IHA response withoutsignificant protection or, conversely, elicit little IHA response butsubstantial protection. In either case, it is not surprising that oralexposure would elicit a good response, assuming that such exposure issufficient to qualify as “prior experience.” The subcutaneousvaccination, while apparently effective, elicited a relatively minor IHAresponse. Our prior experiment in small ruminants using IM injectionresulted in substantial IHA responses to both P. haemolytica serotypes 5and 6. Perhaps the route of exposure directed the former to a primarilycell-mediated response and the latter to a more humoral response.

[0097] Antileukotoxin titers were not impressive in either group, asonly 3 of 10 vaccinated animals seroconverted after vaccination.Antileukotoxin titers were substantial prior to vaccination, however,and may have contributed to a decreased response. These preexistingtiters may have been due to previous colonization by serotype 2 P.haemolytica, the most common commensal P. haemolytica in calves' nasalpassages. Alternatively, it is possible that replication of P.haemolytica after vaccination was not great, perhaps because thebacteria were readily handled by the immune system, and therefore littleantigenic mass of leukotoxin was elaborated. Finally, there is thepossibility that the altered leukotoxin protein, although designed toleave immunodominant epitopes, is not particularly adept at stimulatinga neutralizing response even if it is immunogenic. There is littledoubt, however, that some leukotoxin neutralizing antibody was producedin response to vaccination.

[0098] The multiple large areas of firm lung consolidation inunvaccinated animals at necropsy and the relatively large concentrationof P. haemolytica in those areas indicate an active infection whichspread from the initial site of inoculation. In contrast, the vaccinates(except the 3 with essentially clean, culture-negative lungs) hadrelatively smaller areas of consolidation confined to single lung lobeswhich contained moderately high numbers of P. haemolytica. Other lobesof these animals were either culture-negative or contained low tomoderate numbers of bacteria. These data may indicate that the infectionwas active primarily at the site of inoculation and bacteria were havingdifficulty establishing in other portions of the lung. The cultureresults from tracheal specimens might support that conclusion, since 4of the 6 control animals but none of the vaccinates yielded P.haemolytica from this source, which indicates the infection was not wellcontained in most of the controls.

[0099] The data are clear that both subcutaneous administration and oraladministration of the modified-live vaccine were of significant benefitto animals intratracheally challenged with wild-type P. haemolyticaserotype 1. Manipulation of dosage or use of intramuscular injectionsmight further improve the efficacy of parenterally administeredvaccines.

[0100] The orally administered vaccine was markedly efficacious. Thenecessary dose in this case is likely some threshold level which issufficient to cause colonization of the upper respiratory tract orpalatine tonsils. Although conceivable, it is unlikely the dosage waseffective due to passage into the gastrointestinal system.

[0101] Even 10¹⁰ CFU of P. haemolytica passing into the rumen would be arelatively small number of organisms, and the possibility that thesebacteria could compete against the rumen or intestinal flora andmultiply is remote. Still, if the gut were to respond and there is amucosal immune system link in cattle, one might expect the response tobe beneficial. These possibilities might be investigated usinggenetically marked P. haemolytica such as a rifampicin-resistant strainfor which colonization can be detected with great sensitivity (7, 11).

[0102] The theory behind the oral vaccine is that animals naturallyinfected with P. haemolytica serotype 1 develop resistance to subsequentnasal colonization by serotype 1 organisms. They also develop systemicantibodies against P. haemolyica and, variably, against leukotoxin. Anavirulent organism which is proficient at colonization of nasal passagesor palatine tonsils might elicit similar resistance or resistance topulmonary challenge without the possibility of causing pneumonicpasteurellosis.

[0103] It is even possible that passive protection might occur in somecases by competitive exclusion of virulent P. haemolytica. Delivery bycarriage on feedstuffs is possible because the palatine tonsils sustainlong-term colonization by P. haemolytica (18, 19). These sites also arein the path of incoming feed. Often course feedstuffs such as hay stemsare found within the larger sinuses of the palatine tonsils, indicatingthat exposure to feed is significant.

[0104] We conducted preliminary experiments to test the ability of feedto deliver P. haemolytica to palatine tonsils or nasal passages using arifampicin-resistant strain of P. haemolytica. Calves fed infected feedbecame colonized in both tonsils and in nasal passages.

[0105] In summary, protection against virulent challenge was conferredby subcutaneous or oral administration of a modified-live P. haemolyticavaccine. In this experiment, oral administration elicited greaterantibody responses and slightly greater protection. An additionalpotential benefit of vaccination via feed is that calves would not needto be caught to be vaccinated, thereby reducing stress for both the calfand the operator. A potential caveat is that at least some calves musteat or at least browse through the inoculated feed to become colonized.Calves which do not partake of the feed may later become immune afterexposure to calves which did partake.

EXAMPLE 4 Preliminary Assessment of Safety and Efficacy ofOrally-administered Vaccine for Calves already in Typical MarketingChannels

[0106] This experiment was designed to test the efficacy of anexperimental pulmonary vaccine produced by personnel at Texas A & MUniversity. Within that experiment, and balanced between the groups ofcalves utilized by Texas A & M, was our smaller experiment involving 18head of calves. Our experiment was designed to see if feeding ourvaccine strain to calves in the early stages of typical marketingchannels would result in colonization, elicit an immune response, andpossibly reduce the incidence of shipping fever.

[0107] A field experiment was conducted in the Fall of 1997 with 105steer calves (average 207 kg) procured from local sales barns by anOrder-Buyer in eastern Tennessee. Although the primary objective of theexperiment was to test an experimental vaccine by Texas A&M University,18 calves were fed the in-frame leukotoxin mutant 4 days prior toshipment to a feedlot in Texas about 1600 km away. The day afterpurchase, the calves arrived at an order-buyer barn where they wereear-tagged, vaccinated against clostridia, infectious bovinerhinotracheitis, and parainfluenza-3 virus, wormed with ivermectin, andcastrated by banding. Blood was collected for serum, rectal temperatureswere recorded, and nasal mucus specimens were collected.

[0108] Odd numbered calves were vaccinated with the experimental TexasA&M preparation. Nine odd- and nine even-numbered calves were separatedinto a pen approximately 20′ by 40′ which contained a 12′ feed bunk anda source of fresh water. A suspension of P. haemolytica NADC-D153ΔlktA(100 ml) was poured onto 35 kg of a commercial calf ration (Growena,Ralson Purina, St. Louis Mo.) and 15 kg of fresh grass hay. The bacteriawere grown on 10 Columbia agar plates overnight at 37° C. afterspreading inoculum for confluent growth. Growth was harvested into EBSSto a density approximating 2×10⁹ CFU/ml, and the resulting suspensionwas placed on ice until the calves were penned, whereupon 150 ml wastop-dressed onto the above feed.

[0109] Four days after feeding the vaccine, the calves were loaded ontoa truck and transported to Bushland, Tex., where an experimentalfeedyard is operated jointly by the USDA Agricultural Research Serviceand by Texas A&M University. Upon arrival the next day the calvesappeared exhausted, as is typical of shipping this distance. The calveswere run through the chute and rectal temperatures were recorded. Thecalves were then sorted into 6 groups and allowed to rest overnight. Thenext day, the calves were again run through the chute. Blood and nasalmucus was collected, rectal temperatures were recorded, and weights weretaken. Many of the calves were febrile (over 40° C.) with nasaldischarge and loose stool.

[0110] The protocol called for treating calves for shipping fever withantibiotic on the second consecutive day of fever using tilmicosin(Micotil, Eli Lilly, Indianapolis Ind.). Calves not responding within 2days of treatment were to be treated with long-acting tetracycline(LA-200, Pfizer Inc., New York N.Y.). It was deemed expedient,considering the number of hot calves, to run all calves through thechute daily for 4 days to record all rectal temperatures. Serum, nasalmucus, weights, and rectal temperatures were then collected weekly(counting from the day after arrival) for 4 weeks, as described above.

[0111] The second day after arrival, 55 calves were treated usingtilmicosin. Additional calves were treated subsequently until 22 daysafter arrival, bringing the total number treated to 84% of survivinganimals. Ten total animals died within 4 days of arrival, 6 given theTexas A&M product and 4 non-vaccinates. No animals given the oralvaccine died.

[0112] Postmortem observations revealed fibrinous pneumonia in all tendead animals, and P. haemolytica was recovered from all lungs along withP. multocida in a few lungs. Serotyping of lung isolates revealed that 9calves died of pasteurellosis by serotype 1 and 1 calf by serotype 6. Nostatistically significant differences were noted in morbidity (as judgedby treatment) between the orally-vaccinated, Texas A&M-vaccinated, orcontrol animals (78%, 84%, and 87% respectively). Nor was the differencein mortality significant (11.5% of non-orally-vaccinated versus 0% oforally-vaccinated calves, p>0.05).

[0113] Antibody titers (measured by IHA against serotype 1 P.haemolytica) increased significantly (p<0.01) between samples taken atthe order-buyer barn and those taken on arrival at the feedyard for bothorally-vaccinated and Texas A&M vaccinated calves compared tonon-vaccinates. Overall, the calves gained 29 kg between purchase andthe termination of the experiment after 28 days in the feedyard. Onegroup, orally-vaccinated calves which did not receive the Texas A&Mvaccine, gained significantly more weight than any other group (p<0.01,n=9) at 40.2 kg. All other groups did not significantly differ in thisparameter.

[0114]Pasteurella haemolytica serotype 1 and, to a lesser extent,serotype 6 were recovered from nasal mucus of most calves one or moretimes at the feedyard. The groups did not differ significantly inshedding the organism. Some but not all calves which received theoral-vaccine shed the mutant organism in one or more nasal mucusspecimens during the first week at the feedyard, indicating that theinoculum was sufficient to colonize their upper-respiratory tracts underthese conditions.

[0115] This experiment demonstrates that our experimental oral vaccinecan be delivered in feed at an order-buyer barn prior to shipment to thefeedyard and thereby colonize and elicit an immune response within 1week. Morbidity and mortality in the current experiment were unusuallyhigh. In addition to frequent isolations of P. haemolytica, respiratorycoronavirus and P. multocida isolations were common. The number ofcalves from which coronavirus was isolated was unusually high and mayaccount for the unusually heavy morbidity and the frequent diarrheaobserved. The number of calves requiring retreatment was also unusual,suggesting that bacteria other than P. haemolytica played a significantrole in the outbreak.

[0116] Tilmicosin is an antibiotic with a narrow spectrum of activity,targeted and advertised primarily to combat P. haemolytica. Given thatbacteria other than P. haemolytica and viruses such as respiratorycoronavirus were prevalent, it is not particularly surprising that themonovalent vaccines against P. haemolytica did not significantly reducemorbidity. However, none of the orally-vaccinated calves succumbed topneumonic pasteurellosis compared to 11.5% of the others, suggestingthat the vaccine played a role in reduction of mortality. Thesubstantially greater weight gain of calves given only the oral vaccinefurther supports the conclusion that the vaccine reduced disease inthese calves. Administration of the Texas A&M product together with theoral vaccine may have resulted in a reduction in the response to one orboth products or in responses deleterious to disease-resistance andthereby reduced the benefit conferred by the oral vaccine alone.

EXAMPLE 5 Ability of the P. haemolytica Serotype 1 Leukotoxin In-frameDeletion to Colonize Nasal Passages of Calves Stressed by ConcurrentBovine Herpes Virus Type 1 Infection

[0117]Pasteurella haemolytica serotype 1 is recovered sporadically inrelatively low amounts from nasal mucus specimens of normal healthycalves. After stress or respiratory viral infection, P. haemolyticaserotype 1 can proliferate explosively in nasal passages to become thepredominant flora. Very high amounts of bacteria are shed in nasal mucusof such calves. It is believed that these high numbers of bacteria areinhaled or aspirated into susceptible lung to result in pneumonicpasteurellosis. Thus, this experiment was designed to obtain preliminarydata on whether leukotoxin deletion mutants of P. haemolytica cancolonize nasal passages under these conditions and, if so, whether theymight competitively exclude colonization by wild-type P. haemolytica.Both serotype 1 and serotype 6 organisms were used because both areknown to cause fatal fibrinous pneumonia in calves.

Materials and Methods

[0118] Vaccination of animals. Eight crossbred dairy-type calves, about150 kg, were purchased from a local dairy and maintained at the NADC.The calves were separated randomly into 2 groups of 4 each such that nocontact could occur between the groups. The calves were allowed toacclimate for 10 days prior to initiation of the experiment.

[0119] Infectious bovine rhinotracheitis virus (Coopers strain, kindlyprovided by National Veterinary Services Laboratories) was aerosolizedinto each calf's nostrils on inspiration, according to instructionsprovided by NVSL for challenge, resulting in a final dosage of 10⁹ ⁴TCID₅₀ /nostril. After exposure to virus, one group of 4 calves were feda palatable feed concentrate onto which 10 ml/calf of a mixed suspensionof P. haemolytica D153 ΔlktA and D174ΔlktA (serotypes 1 and 6respectively) at 2×10⁹ total CFU/ml was poured. The other group was feduninoculated ration.

[0120] Five days after exposure to virus, the fed group was exposed byintranasal injection to 1.5 ml/nostril of P. haemolytica (mixture asabove) at 2.7×10⁸ total CFU/ml. Six days after exposure to virus, allgroups were exposed by intranasal injection to a mixture of wild-type P.haemolytica D153 and D174 at 5×10⁸ total CFU/ml.

[0121] Sample collection and analysis. Nasal mucus specimens werecollected on the day of exposure to virus, and 3, 4, 5, 6, 7, and 10days after virus exposure. Serum was collected the day of exposure and10 days later.

[0122] On the tenth day after exposure to virus, all calves wereeuthanized, and the lungs were examined grossly. Rectal temperatureswere recorded daily from the day of exposure to virus until euthanasia.Serum was tested for antibody against both serotype 1 and serotype 6 P.haemolytica by IHA.

[0123] Nasal mucus was diluted in 10-fold increments and spread ontoblood agar base plates containing 5% defibrinated bovine blood. Afterovernight incubation, P. haemolytica were identified and enumerated, and20 representative colonies were serotyped by a rapid plate agglutinationprocedure.

Results

[0124] Most calves were febrile within 3 days of virus-exposure, andpeak fevers occurred on day 4 at 40.5° C. Only 3 calves remained febrilemore than one week, and all became afebrile by 10 days after virusexposure.

[0125] All calves were culture-negative for P. haemolytica in nasalmucus the day of virus-exposure. One calf fed P. haemolytica leukotoxinmutants shed non-hemolytic serotype 1 organisms in its nasal mucusspecimens starting 3 days after virus-exposure and continued to shedleukotoxin mutants until euthanasia. The remaining 3 fed calves remainedculture-negative for P. haemolytica until day 6, one day afterintranasal exposure to the mixture of leukotoxin mutants. These calvesshed non-hemolytic P. haemolytica on days 6, 7, and, with one exception,day 10 (Table 5). The calves not deliberately exposed to P. haemolyticauntil day 6 remained culture negative for the organism until day 7,whereupon they shed mixtures, with one exception on day 10, of serotype1 and serotype 6 hemolytic P. haemolytica.

[0126] Three animals exposed to mutant P. haemolytica seroconverted(4-fold or greater increase in titer) to both serotypes 1 and 6 betweenthe time of virus-exposure and euthanasia. The fourth animal had atwo-fold titer increase against both serotypes. The remaining animalseither increased 2-fold or maintained a constant titer during thatperiod.

[0127] The lungs at postmortem were mostly unremarkable. Calf 30,unexposed to leukotoxin mutants, had firm consolidation throughout itsright caudal half of the middle lobe with 5% involvement of the cranialhalf. Calves 17 and 18 had minor lesions of consolidation involving 5%or less of 2 and 3 lung lobes respectively. No abnormalities were notedin the remaining calves.

[0128]Pasteurella haemolytica leukotoxin mutants were capable ofcolonizing the nasal passages of calves which were concurrently infectedwith IBR virus. Such colonization did not prevent or even reduceexperimental superinfection with wild-type P. haemolytica. Judging bynumbers of P. haemolytica shed in nasal mucus, it appears the leukotoxinmutants were less robust in nasal colonization. The wild-type bacteriacolonized at levels about 10-fold higher than did the mutants whether bythemselves or together. Nevertheless, the leukotoxin mutants were ableto maintain a substantial level of colonization even in the presence ofwild-type P. haemolytica, indicating that the bacteria were still quiterobust. In fact, mixtures of wild-type parent strains and leukotoxinmutants passed in vitro in Columbia broth for 100 generations resultedin a population slightly enriched for leukotoxin mutants, indicatingthat the leukotoxin mutants compete very well with wild-type under thoseconditions. Whether it is possible to superimpose infection withleukotoxin mutants in the face of substantial colonization by wild-typeP. haemolytica is not known. Perhaps the leukotoxin mutants maintainedtheir infection because they already had a foothold in the nasopharynx.

[0129] Our previous work with P. haemolytica infections using an IBRvirus model indicates that bacterial infection of the nasopharynx(specifically, the palatine tonsils) does not necessarily translate intoexplosive colonization of the nasal passages. Some calves which wereknown carriers of P. haemolytica serotype 1 in the palatine tonsilsfailed to become colonized in the nasal passages even though the nasalpassages were susceptible, as demonstrated by intranasal inoculation.Other similar calves of probable but unconfirmed carrier status didbecome colonized under similar conditions. This seeming paradox,infection in the pharynx which may or may not extend into adjacentsusceptible nasal passages, is not easy to explain. Perhaps the ciliaryflow from nasal passages carries material both forward, out of thenares, and backwards into the oropharynx.

[0130] Serum antibody titers against both serotype 1 and serotype 6increased substantially in three of the calves fed leukotoxin mutants.Since the calves were killed on day 10, little time was available for animmune response in the 7 calves which did not colonize until day 6 or 7.It is therefore likely that feeding the organism elicited or at leastfacilitated and immune response prior to the detected nasalcolonization.

[0131] Both serotypes 1 and 6 were recovered from nasal mucus in highamounts from every calf, most often as a mixed infection with bothserotypes. In two cases, by day 10 serotype 1 had outgrown serotype 6 tobecome the predominant flora. In one case, serotype 6 became thepredominant flora. These results suggest that serotype 6 is nearly equalin its ability to colonize under the chosen conditions. Givenobservations that serotype 6 strain NADC D174 elicits severe pneumoniain calves after intratracheal inoculation, one would expect thatrespiratory disease would occur in calves under conditions in the field.In fact, serotype 6 P. haemolytica has been recovered previously fromnasal passages of calves in field trials and from lungs of calves whichsuccumbed to pneumonic pasteurellosis. While serotype 1 remains the mostcommon isolate in both nasal passages of stressed calves and frompneumonic lung, serotype 6 makes up a significant percentage of P.haemoytica isolations from nasal mucus or lung (about 10%) under theseconditions.

[0132] Thus, in-frame leukotoxin deletion mutants of P. haemolytica arecapable of colonizing the nasopharynx of calves made susceptible withconcurrent IBR virus infection. Such infection was not sufficient toprevent colonization by wild-type P. haemolytica. Feeding the leukotoxinmutants to calves concurrently with IBR virus exposure allowed one calfto become colonized to a high level in its nasal passages and appearedto result in seroconversion to P. haemolytica in 3 of 4 calves. Both P.haemolytica serotypes 1 and 6 are capable of explosive colonizationduring respiratory virus infection, and each can do so in the presenceof the other. TABLE 1 IHA antibody titers against Pasteurellahaemolytica serotypes 5 and 6 and leukotoxin neutralization titersbefore and after vaccination. First dose of vaccine on day 0, 2^(nd)doseon day 21. All animals intratracheally challenged with wild-typeserotypes 5 and 6 on day 28. n = 5 per group. Day 0 Day 14 Day 28 Day 33Serotype 5 Vaccinate 2.4 6.0 6.2 6.8 Control 1.2 1.8 2.6 8* Serotype 6Vaccinate 1.4 6.2 6.2 6.8 Control 0.8 1.4 1.4 6* Leukotoxin** Vaccinate0.4 3.0 3.4 —

[0133] TABLE 2 Lung lesion scores and postmortem lung bacterial cultureresults 5 days after intratracheal challenge with Pasteurellahaemolytica serotypes 5 and 6. (n = 5 for each group, numbers expressed± 95% confidence interval) Geometric mean P. Percent lung lesions**haemolytica in lung Vaccinates  3.5 ± 2.8* 1.2 x 10¹ ± 0.9 x 10¹*Controls 52.1 ± 21.7  6.3 x 10⁷ ± 2.5 x 10¹ 

[0134] TABLE 3 IHA antibody titers against Pasteurella haemolyticaserotype 1 and leukotoxin neutralization titers before and aftervaccination. First dose of vaccine on day 0, 2^(nd)dose on day 21. Allanimals intratracheally challenged with wild- type serotype 1 on day 28.(n = 6 for controls and 5 each for vaccinated groups) Day −3 Day 21 Day28 Day 32 or 33 IHA titer IM vaccinate 2.6 3.4 4.8 5.8 Oral 3.0 7.6 7.07.6 vaccinate Control 2.2 3.3 3.5 5.8* Leukotoxin** IM vaccinate 6.8 6.87.4 7.8 Oral 6.6 7.8 7.4 8.0 vaccinate Control 6.8 6.3 6.2 6.8*

[0135] TABLE 4 Lung lesion scores and postmortem lung bacterial cultureresults 4 or 5 days after intratracheal challenge with Pasteurellahaemolytica serotype 1. (n = 6 for controls and 5 each for vaccinatedgroups, numbers expressed ± 95% confidence interval) Geometric meanPercent lung lesions*** P. haemolytica in lung IM vaccinate  7.0 ± 7.3*1.8 × 10² ± 0.7 × 10²* Oral vaccinate  4.4 ± 4.5** 1.4 × 10² ± 0.6 ×10²* Controls 32.0 ± 13.4 1.6 × 10⁶ ± 1.0 × 10²

[0136] TABLE 5 Shedding of P. haemolytica in nasal mucus of calvesinfected with IBR virus on day 0. Day 6 Day 6 Day 10 Calf* Phenotype**CFU/ml % St-1† CFU/ml % St-1 CFU/ml % St-1 15 mutant 4.0 × 10⁷ >95 4.0 ×10⁶ 50 1.0 × 10⁸ >95 wild-type none — 1.5 × 10⁸ >95 2.0 × 10⁸ 80 19mutant 5.6 × 10⁷ 85 1.1 × 10⁷ 65 none — wild-type none — 1.1 × 10⁸ 105.0 × 10⁸ <5 28 mutant 4.3 × 10⁷ 80 2.5 × 10⁷ 70 1.2 × 10⁷ 60 wild-typenone — 1.2 × 10⁸ 55 1.9 × 10⁸ 20 29 mutant 1.6 × 10⁷ >95 2.0 × 10⁶ >954.0 × 10⁶ >95 wild-type none — 6.0 × 10⁷ 15 1.3 × 10⁸ >95 5 wild-typenone — 2.0 × 10⁸ 60 1.5 × 10⁸ 60 17 wild-type none — 1.5 × 10⁸ 50 4.1 ×10⁷ 40 18 wild-type none — 2.0 × 10⁸ 10 2.0 × 10⁸ >95 30 wild-type none— 2.8 × 10⁸ 30 6.0 × 10⁸ 30

References

[0137] 1. Briggs R. E., Tatum F. M., Casey T. A., Frank G. H.Characterization of a restriction endonuclease, PhaI, from Pasteurellahaemolytica serotype A1 and protection of heterologous DNA by a clonedPhaI methyltransferase gene. Appl. Environ. Microbiol. 60:2006-2010.1994.

[0138] 2. Thomas C. M. Plasmid replication. In: PLASMIDS: A PRACTICALAPPROACH. K. G. Hardy, ed. IRL Press Limited, Oxford, England. 1987.

[0139] 3. Tatum F. M., Briggs R. E., Sreevatsan S. S., Zehr E. S., LingHsuan S., Whiteley L. O., Ames T. R., Maheswaran S. K. Construction ofan isogenic leukotoxin deletion mutant of Pasteurella haemolyticaserotype 1: characterization and virulence. Microb. Pathog. 24: 37-46,1998.

[0140] 4. Conrad M., Topal M. D. Modified DNA fragments activate NaeIcleavage of refractory DNA sites. Nucleic Acids Res; 20:5127-5130. 1992.

[0141] 5. Murphy G. L., Whitworth L. C., Clinkenbeard K. D.,Clinkenbeard P. A. Hemolytic activity of the Pasteurella haemolyticaleukotoxin. Infect. Immun. 63:3209-3212. 1995.

[0142] 6. Fedorova N. D., Highlander S K. Generation of targetednonpolar gene insertions and operon fusions in Pasteurella haemolyticaand creation of a strain that produces and secretes inactive leukotoxin.Infect. Immun. 65:2593-2598. 1997.

[0143] 7. Briggs R. E., Frank G. H., Zehr E. S. Development and testingof a selectable challenge strain of Pasteurella haemolytica for studiesof upper-respiratory colonization of cattle. Am. J. Vet. Res. 59:401-405, 1998.

[0144] 8. Frank G. H., Smith P. C. Prevalence of Pasteurella haemolyticain transported calves. Am. J. Vet. Res. 44:981-985. 1983.

[0145] 9. Frank G. H., Wessman G. E. Rapid plate agglutination procedurefor serotyping Pasteurella haemolytica. J. Clin. Microbiol. 7:142-145.1978.

[0146] 10. Frank G. H., Briggs R. E., Loan R. L., Purdy C. W., Zehr E.S. Serotype-specific inhibition of colonization of the tonsils andnasopharynx of calves by Pasteurella haemolytica serotype A1 aftervaccination with the organism. Am. J. Vet. Res. 55: 1107-1110. 1994.

[0147] 11. Frank G. H., Briggs R. E., Zehr E. S. Colonization of thetonsils and nasopharynx of calves by a rifampicin-resistant Pasteurellahaemolytica and its' inhibition by vaccination. Am. J. Vet. Res. 56:866-869. 1995.

[0148] 12. Frank G. H., Briggs R E., Loan R. W., Purdy C. W., Zehr E. S.Respiratory tract disease and mucosal colonization by Pasteurellahaemolytica in transported cattle. Am. J. Vet. Res. 57: 1317-1320. 1996.

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[0150] 14. McVey D. S., Loan R. W., Purdy C. W., Richards A. E.Antibodies to Pasteurella haemolytica somatic antigens in two models ofthe bovine respiratory disease complex. Am. J. Vet. Res. 50:443-447.1989.

[0151] 15. Jones G. E., Donachie D. W., Sutherland A. D., Knox D. P.,Gilmour J. S. Protection of lambs against experimental pneumonicpasteurellosis by transfer of immune serum. Vet. Microbiol. 20:59-71.1989.

[0152] 16. Schimmel D., Erler W., Diller R. [The significance ofantibodies to Pasteurella haemolytica A1 in the colostrum of cows andblood serum of calves]. Berl Munch Tierarztl Wochenschr 105:87-89.1992.

[0153] 17. Frank G. H., Briggs R. E. Colonization of the tonsils ofcalves with Pasteurella haemolytica. Am. J. Vet. Res 53:481-484. 1992.

[0154] 18. Frank G. H., Briggs R. E., and Debey B. M. Bovine tonsils asreservoirs for Pasteurella haemolytica : Colonisation, immune response,and infection of the nasopharynx. In: Pasteurellosis in ProductionAnimals (Workshop Proceedings, Australian Centre for InternationalAgricultural Research.) pp 83-88. 1992.

1 2 1 26 DNA Pasteurella cf. haemolytica 1 ccggatcccc aattcgtaga ggtttc26 2 26 DNA Pasteurella cf. haemolytica 2 ccggatccgc tgaaagcggt cggggg26

We claim:
 1. A P. haemolytica bacterium which: a) expresses no biologically active leukotoxin, b) expresses a form of leukotoxin molecule which induces antibodies which specifically bind to leukotoxin; and c) contains no foreign DNA.
 2. The P. haemolytica bacterium of claim 1 wherein the form of leukotoxin molecule expressed is a deletion mutant.
 3. The P. haemolytica bacterium of claim 2 wherein the deletion mutant is about 66 kD.
 4. The P. haemolytica bacterium of claim 2 wherein the deletion mutant lacks amino acids 34 to
 378. 5. The P. haemolytica bacterium of claim 1 wherein the bacterium is lkt C⁺.
 6. The P. haemolytica bacterium of claim 1 wherein the leukotoxin operon comprises no antibiotic resistance genes.
 7. The P. haemolytica bacterium of claim 1 which comprises a mutation in the structural gene lktA⁻which encodes leukotoxin.
 8. The P. haemolytica bacterium of claim 1 wherein the bacterium comprises a mutation which is non-reverting, said mutation resulting in the inability of the bacterium to express biologically active leukotoxin.
 9. A method of inducing immunity to pneumonic pasteurellosis in ruminants, comprising the step of: administering the bacterium of claim 1 to a ruminant whereby immunity is induced.
 10. The method of claim 9 wherein the step of administering is via the oral route.
 11. The method of claim 10 wherein the bacterium is top-dressed on the feed of the ruminant.
 12. The method of claim 9 wherein the step of administering comprises injecting the bacterium subcutaneously.
 13. The method of claim 9 wherein the step of administering comprises injecting the bacterium intradermally.
 14. The method of claim 9 wherein the step of administering comprises injecting the bacterium intramuscularly.
 15. The method of claim 9 wherein the step of administering is via the nose.
 16. A feed for ruminants which comprises the bacterium of claim
 1. 17. A vaccine for reducing morbidity in ruminants, comprising: a P. haemolytica bacterium which: a) expresses no biologically active leukotoxin, b) expresses a form of leukotoxin molecule which induces antibodies which specifically bind to leukotoxin; and c) contains no foreign DNA.
 18. A temperature sensitive plasmid which replicates at 30° C. but not at 40° C. in P. haemolytica and which has an origin of replication of the same incompatibility group as the plasmid which has been deposited at the ATCC with Accession No. ______.
 19. The temperature sensitive plasmid of claim 16 which is the plasmid which has been deposited at the ATCC with Accession No. ______.
 20. A P. haemolytica leukotoxin molecule which: a) is biologically inactive; b) induces antibodies which specifically bind to leukotoxin; and c) contains no foreign amino acid sequences.
 21. The P. haemolytica leukotoxin protein of claim 20 wherein the form of leukotoxin molecule expressed is the result of a deletion mutation.
 22. The P. haemolytica leukotoxin protein of claim 21 wherein the protein is about 66 kD.
 23. The P. haemolytica leukotoxin protein of claim 21 wherein the protein lacks amino acids 34 to
 378. 24. The P. haemolytica leukotoxin protein of claim 20 wherein the leukotoxin protein is acylated.
 25. The P. haemolytica leukotoxin protein of claim 20 wherein the leukotoxin protein comprises no antibiotic resistance enzymes.
 26. A method of inducing immunity to pneumonic pasteurellosis in ruminants, comprising the step of: administering the leukotoxin protein of claim 20 to a ruminant whereby immunity is induced.
 27. The method of claim 26 wherein the step of administering is via the oral route.
 28. The method of claim 26 wherein the leukotoxin protein is top-dressed on the feed of the ruminant.
 29. The method of claim 26 wherein the step of administering comprises injecting the leukotoxin protein subcutaneously.
 30. The method of claim 26 wherein the step of administering comprises injecting the leukotoxin protein intradermally.
 31. The method of claim 26 wherein the step of administering comprises injecting the leukotoxin protein intramuscularly.
 32. The method of claim 26 wherein the step of administering is via the nose.
 33. A feed for ruminants which comprises the leukotoxin protein of claim
 20. 34. A vaccine for reducing morbidity in ruminants, comprising: a P. haemolytica leukotoxin protein which: a) is biologically inactive; b) induces antibodies which specifically bind to leukotoxin; and c) contains no foreign amino acid sequences. 